The invention relates to the three-dimensional structure of a crystal of a kinase enzyme complexed with a ligand. The three-dimensional structure of a protein kinase-ligand complex is disclosed. The invention also relates to methods of preparing such crystals. Kinase-ligand crystal structures wherein the ligand is an inhibitor molecule are useful for providing structural information that may be integrated into drug screening and drug design processes. Thus, the invention also relates to methods of using the crystal structure of kinase enzyme-ligand complexes for identifying, designing, selecting, or testing inhibitors of kinase enzymes, such inhibitors being useful as therapeutics for the treatment or modulation of i) diseases; ii) disease symptoms; or iii) the effect of other physiological events mediated by kinases; having one or more kinase enzymes involved in their pathology.
T-cell activation is a complex process that results from the integrated activation of multiple signal transduction pathways [1-3]. One of the earliest T-cell signaling events observed upon T-cell receptor (TCR)-ligand engagement is the CD4/CD8-dependent activation of lymphocyte kinase (Lck), a member of the non-receptor Src family of tyrosine kinases [4-8]. Lck phosphorylates and activates a number of substrates necessary for TCR signaling [9]. Perhaps the best understood activity of Lck is the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the TCR xcex6-subunit [4, 6, 9]. The extent of xcex6-chain ITAM phosphorylation dictates the threshold for ligand-mediated TCR signaling and T-cell activation [10, 11]. Phosphorylated ITAMs serve as high affinity docking sites for the recruitment of additional signaling factors, particularly the Syk family tyrosine kinase ZAP-70 [12, 13]. Dual phosphorylation of tyrosines in the ITAMs by Lck is required for the binding of tandem ZAP-70 Src homology-2 (SH2) domains [14-16]. Co-localization of ZAP-70 and Lck to the TCR-xcex6 subunit-CD4/8 complex facilitates the Lck-mediated activation of ZAP-70 and subsequent ZAP-70 autophosphorylation [17-21]. Activated Lck and ZAP-70 perpetuate the TCR signaling cascade by providing additional docking sites for other SH2 containing kinases (including Fyn, Syk and Itk), adaptor proteins (including SLP-76, SHC, LAT, FyB and Grap), and transducing elements (including PLCxcex3, PI3-kinase and Rac/Rho) [2, 3, 22]. Biochemical information is then transmitted down multiple signaling pathways, including the Ras/mitogen-activated protein kinase pathway, the phosphatidylinositol pathway, and the Rho/Rac pathway [2]. Among other effects, TCR signaling up-regulates transcription and translation of IL-2 and IL-2 receptors which are prerequisites for T-cell proliferation.
Genetic studies have demonstrated that Lck expression is restricted to lymphocytes. Loss of Lck expression in human Jurkat T-cells results in a loss of signaling in response to TCR ligation [23, 24]. In addition, inactivation of the Lck gene, or expression of dominant negative transgenes in mice, results in early arrest of thymocyte maturation [25-27]. These and other biochemical studies have implicated Lck as an essential early mediator of the TCR signaling pathway. Lck therefore represents an attractive target for therapeutic intervention in T-cell mediated disorders such as autoimmune diseases and transplant rejection.
Lck is a modular protein consisting of a C-terminal catalytic domain, a single Src homology-2 (SH2) and a Src homology-3 (SH3) domain, and a unique N-terminal region. The N-terminal region is involved in anchoring Lck to CD4/8 through Zn2+ coordination with conserved cysteine residues present in both proteins [28, 29]. The activity of Lck is regulated by autophosphorylation of Tyr-394 located in the catalytic domain activation loop [30] and by the phosphorylation of Tyr-505 by C-terminal Src kinase (Csk) [31-33]. Further understanding of the regulation of Lck has been provided by the crystal structures of two other Src family protein kinases, c-Src and Hck [34-36]. From these structures it can be delineated that the SH2 and SH3 domains function in part to negatively regulate Lck activity by forming intramolecular contacts that stabilize the catalytic domain in an inactive conformation [37]. The SH2 domain binds to phosphorylated Tyr-505 and the SH3 domain associates with a proline containing motif in a hinge region connecting the SH2 and catalytic domains [34-36]. Release of these intramolecular regulatory constraints by dephosphorylation of Tyr-505 [38] and/or the presence of competing SH3/SH2 ligands [39] results in the autophosphorylation of Tyr-394 in the activation loop and a catalytically active kinase [19]. A structural basis for Lck activation has been previously elucidated from the crystal structure of an autophosphorylated Lck catalytic domain [40].
Protein kinases have been implicated as potential targets for a variety of clinical applications. The identification of molecules, such as inhibitors, that bind to kinase enzymes, affect kinase activity and thereby influence pathological processes, is valuable for investigating potential therapeutics for disease, or disease symptoms, that are mediated by kinase enzymes. Such identification has been attempted using methods such as the screening of large numbers of random libraries of natural and/or synthetic compounds, hoping that some number of random compounds will demonstrate the desired biological activity. This method is inefficient in that it typically results in a small number of xe2x80x9chitsxe2x80x9d and it is constrained by the limitations imposed in actually screening large numbers of compounds in laboratory assays. An improved method of such identification is structure-based drug design (xe2x80x9cSBDDxe2x80x9d). SBDD comprises a number of integrated components, including, structural information (e.g., spectroscopic data such as X-ray or magnetic reasonance information, relating to enzyme structure and/or conformation, enzyme-ligand interactions, etc.), computer modeling, medicinal chemistry, and biological testing (both in vitro and in vivo). These components, each alone and in combination, are useful for accelerating the drug discovery process, for gaining insight into disease and disease processes, and for providing a more efficient method for identifying drug candidates.
Efforts to understand the molecular constraints necessary to achieve inhibitor potency and selectivity have been aided by an increasing number of crystal structures of different protein kinases complexed with ATP-competitive inhibitors. One such inhibitor is staurosporine, an alkaloid that has been previously shown to inhibit a broad range of tyrosine and serine/threonine kinases with nanomolar potency [41]. Crystal structures of staurosporine bound to the serine/threonine kinases protein kinase A (PKA) and the cyclin-dependent kinase 2 (CDK2) elucidated the binding mode of this inhibitor to protein kinases [42, 43] (reviewed in [44]). A similar binding mode has been reported in a recently solved structure of the tyrosine kinase Csk in complex with staurosporine [45]. Described herein are crystal structures of Lck complexed with staurosporine obtained from both soaking and co-crystallization experiments. Comparison of these two complexes and those previously reported further elucidates the structural basis for the high potency and poor selectivity of this inhibitor.
To date, the three-dimensional structures of Hck/AMP-PNP and Hck/Quercetin complexes have been reported, however, these ligands are not src-selective ligands. The three-dimensional structure of c-Src (apo form) has been elucidated, however, this structure lacks a ligand bound to the enzyme and therefor lacks critical information regarding the interaction of a ligand with the active site of the enzyme.
The role of Src family members Lck and Fyn in TCR activation has been studied with two related Src kinase inhibitors, PP1 and PP2 [51]. PP1 and PP2 are reported to se inhibit Lck and c-Src in vitro at concentrations much lower than is required to inhibit Zap-70, JAK2, EGF-R kinase and protein kinase A [51]. These compounds also inhibit anti-CD3-induced protein tyrosine phosphorylation and subsequent IL-2 gene activation in T lymphocytes [51]. Thus, it appears that PP1 and PP2 dissect a component of TCR signaling not distinguished by other immunosuppressive drugs such as cyclosporin and FK-506. The structural basis for the potency and selectivity of these compounds with the crystal structure of PP2 bound to Lck is described herein. This structure is a useful tool in the design of specific Lck inhibitors and aids in the tailoring of inhibitors, such as PP1 and PP2, to enhance their physical properties, including their therapeutic and pharmaco-kinetic properties.
There is a need for three-dimensional structures of kinase-ligand complexes in order to garner a better understanding of the important interactions between a kinase and its bound ligand, and to utilize this information for methods to identify, design and test molecules with improved binding affinity and molecules that would be useful as therapeutics and/or modulators of kinase-mediated physiological events.
The present invention provides crystals of kinase-ligand complexes suitable for X-ray diffraction analysis. The invention also relates to methods for preparing the crystals of kinase-ligand complexes, particularly where the ligand is an inhibitor of the kinase enzyme. The invention also relates to the detailed three-dimensional structural information of the protein-ligand complexes constituting these crystals, and use of the structure coordinates to reveal atomic details of the active site(s) and other physicochemical interactions that enhance interaction and/or association between the kinase and the ligand. It is also an object of this invention to use the kinase-ligand complex crystals, the three-dimensional structural information provided by the kinase-ligand complex crystals, and the structure coordinates of the kinase-ligand complex in methods to identify, design, select, and evaluate potential inhibitors of kinases that would be useful as therapeutics for diseases or symptoms of diseases that are associated with kinase-mediated physiological events. Such methods may also include use of computer modeling of potential inhibitors based on the the kinase-ligand complex crystals, the three-dimensional structural information of the kinase-ligand complex crystals, and the structure coordinates of the kinase-ligand complex crystals.